Certain issues in modem pharmacological industry involve counteracting two medical-biological problems:
forming a resistance (tolerance and pharmacological efficacy decrease) to medicinal agents including cases due to activation of the MDR-genes system; and PA1 forming of undesirable resorptive effects manifested, first of all, by alteration of the immunocompetent cell system and hemopoiesis; cardio-, hepato-, nephro- and neurotoxicity. PA1 a) Inducting production of a wider range of cytokine and hemopoietic factors that can determine the presence of largely modulating effects rather than only stimulating ones. PA1 b) Reproduction of particular cytokine effects, for instance, IL-2.
A typical cause for these problems is a wide administration of chemotherapeutic agents that can be quite effective, according to their physical-chemical properties, but can also be foreign to an organism at their very nature. Even genetic-engineering medicines, in spite of human genes (DNA) application as a matrix for multiplication, use single-celled organisms (Escherichia coli, yeast cells) that bring in their own, and therefore, xenobiotic contribution into obtained drugs.
Theoretical and practical medicinal research are now paying greater attention to natural key metabolites, i.e., key factors (low molecular biochemical substances) that are naturally determined to trigger chain reactions for endogenous production and modification of many biologically active products for physiologically important and adequate processes. In some cases these biochemical substances function as "biochemical gyroscopes" assigned to restoring the balance of altered equilibrium of basic metabolic processes, for example, anabolism and catabolism or proliferation and differentiation. Alteration of the balance of these vital mechanisms can lead to cell destruction (cytolytic syndrome) or their transformation into malignant ones, i.e., cancer. As a rule, the key regulatory metabolites, i.e., "cellular hormones", are peptide origin factors (usually not larger than 3-20 amino acids). Obtaining synthetic analogues (biochemical substances) and, therefore, drugs with predetermined properties is a desired goal because these drugs are optimal as metabolic therapy instruments and, in fact, are not foreign to the organism.
Ideal peptide structures that can function as these synthetic analogues include sulphur-containing peptides and derivatives thereof, due to the presence of a thiol group. In particular, biological effects of the tripeptide "reduced glutathione" (.gamma.-glutamyl-cysteinyl-glycine; hereinafter--GSH) are known to be researched extensively. The glutathione tripeptide dimer, oxidized glutathione (.gamma.-glutamyl-cysteinyl-glycine; hereinafter--GSSG), where two molecules of the tripeptide with the aforementioned formula are linked via a covalent bond between cysteine residues, is also well known.
Administration of the exogenous synthetic GSSG analogue is known to induce cytokine and hemopoietic factor synthesis during in vitro experiments, and to provide the setting of the cytokine profile to normal values in conditions of cyclophosphamide and radiation immunodepression models (in vivo experiments) along with the immunity and hemopoietic system restoration (International application WO 97/21444, MKI A61.kappa.38/02, published Jun. 19, 1997).
In their turn, the exogenous GSSG drug forms applied to severe immunodeficient conditions and suppressed bone marrow hemopoiesis containing chemical examples relating to clinical Examples in the International Application on AIDS patients, patients with oncopathology, aplastic anemia,and other conditions at treatment courses of different durations, can provide a curative effect in restoring the organism immune status (including antitumor immunity indices), immunogenesis and hemopoietic functional activity (International application WO 97/21444, MKI A61.kappa.38/02, published Jun. 19, 1997).
Previously, the thiol class of biologically active substance had applications aimed to provide GSH at increased levels, i.e., to obtain antioxidant effects. In addition, pharmacological activity was observed, namely, for the pro-oxidant effect gaining and forming of a new intracellular redox-balance by introduction of GSSG that possesses pro-oxidant potential into an organism. This is the only known case of triggered redox-sensitive mechanisms for immunologically significant genes, activation of the cellular thiol metabolism, and therefore, beneficial pharmacological properties were provided including systemic cell-protective effects and immunity state regulation depending on initial cell status: immunodeficiency, i.e., hyporeactivity, immunoautoaggression, i.e., hyperreactivity.
International Application WO 97/21444, MKI A61.kappa.38/02, published Jun. 19, 1997, is directed to gain a set of technical and pharmaceutically acceptable solutions effective for the prevention of the GSSG reduction into GSH and, thus, for extending the lifetime of GSSG as the oxidized form in biological media. Attainment of the biological-pharmacological effects of the glutathione oxidized form is proven by the biomedical investigation results obtained in the course of the complex and extensive preclinical and clinical studies program on synthetic GSSG analog effectiveness. A strategy for extending the lifetime of oxidized GSSG includes providing: salts thereof, composite drugs including GSSG combinations with substances that prolong or enhance the effect of GSSG or salts thereof, or derivatives as a new composite, i.e., a mixture of oxidized GSSG and other materials. The pharmaceutically acceptable GSSG derivative in the form of the salts thereof, or combinations with extenders of the GSSG existence in the oxidized form, or GSSG combinations with enhancers/modifiers, i.e., all technical solutions stabilizing in the varying degree the GSSG molecule disulfide form were first demonstrated to be significantly more effective in inducing the cytokine and hemopoietic factor production in normal conditions and to a greater degree in pathologic ones.
GSSG derivative drug forms are characterized with a larger fraction of the GSSG stabilized in the disulfide form, with maximal pharmacokinetics in biological media. These forms manifested the following features:
The events developing in cells (tissues and, therefore, organs) after interaction with cytokines is well known. These events are conditioned by the universal cytokine influence on the main signal-transducing systems and, through the latter, on the cell genome determining regulating cytokine effects on proliferation, differentiation, and apoptosis.
Methods for obtaining the oxidized GSSG form from the reduced GSH precursor are well known. The labile mercapto-SH-groups of cysteine in GSH can be oxidized with such soft oxidizers as air oxygen (R. Douson, D. Elliot, W. Elliot, K. Jones. Biochemist's manual, Moscow, "Mir", 1991; Tam J. P. et al., Int. J. Pept. Prot. Res., Vol. 29, p. 421-431, 1987; Ahmed A. K. et al., J. Biol. Chem. 250, p. 8477-8482, 1975). However, the reaction rate is rather low in this case and desired quantitative yields require very long periods of time (many days). Catalysis by heavy metals ions and, especially, by copper can be toxic, and can create significant problems for obtaining pure pharmaceutical medicines.
Another oxidation method involves more potent oxidizers such as, hydrogen peroxide, iodine, potassium ferrocyanide, etc. (Kamber B. et al., Helv. Chim. Acta., Vol. 63, p. 899-915, 1980; Hope D. B. et al., J. Biol. Chem., Vol. 237, p. 1563-1566, 1962). These reactions generally proceed much faster (dozens of minutes to several hours). A disadvantage, however, is a difficulty in controlling reaction conditions that can result in significant contamination of the product with oxidation products, e.g., derivatives of the corresponding acids. It is sometimes necessary to add additional, sometimes rather labor-consuming purification procedures, that can sharply appreciate the process.
Yet another oxidation method involves the use of gaseous substances (nitrogen oxides), sulfoxides and other compounds as oxidizers. These oxidizers, however, can be toxic. (William A. Kato et al., Chem. Pharm. Bull., vol. 34 (2), p. 486-495, 1986; A. Meister et al., Ann. Rev. Biochem., p. 711-718, 1983.)
All these methods do not necessarily improve the quantitative yield of the desired product compared to older methods, and at the same time, can provide additional problems regarding toxicity and safety, as in case of nitrogen oxides, or more difficult accessibility of the reagents and their high cost.
Another previously known method involves the use of hydrogen peroxide as an oxidizer (Amber B. et al., Helv. Chim. Acta., Vol. 63, p. 899-915, 1980). The process is performed in the water solution with pH about 8.0-8.5 using the hydrogen peroxide equivalent at the room temperature. The reaction time is about 1 hour and the product yield is 90%. The main impurities (up to 10%) are other oxidation products, which can be removed only by means of an expensive preparative HPLC separation that can sharply increase the drug cost.